| Plasmid Quickpreps | ||
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| Reference: Sambrook et.al.,1989 | Last updated: 1/27/00 | By: Kay Schneitz |
- grow up single colony in 1.5 ml LB/antibiotic ovn @37°C
- pour into tube, spin for 30 sec
- decant supernatant and resuspend pellet in 100 ml lysis solution
- add 200 ml alkaline SDS, vortex well
- incubate for 5 min on ice
- add 150 ml high salt solution, vortex well
- incubate for ca. 10 min on ice
- spin for 10 min
- remove 400 ml supernatant and transfer to new tube prefilled with 400 ml isopropanole
- vortex well and keep tube on ice for 10 min
- spin for 10 min
- wash pellet with 70% EtOH
- vacuum dry pellet for 5 min and take up in 100 ml 1x TE/20 mg/ml RNAse A
- usually 1-2 ml is enough for digest (high-copy plasmid), keep DNA frozen at -20°C
- phenolize important preps if to be kept for a longer period of time (more than 4-6 months)
Solutions: | ||
lysis solution: 2 ml 50% glucose 0.2 ml 0.5 M EDTA 0.25 ml 1 M Tris-HCl pH 8 ------ 10 ml total | alkaline SDS: 7.6 ml H2O 2 ml 5% SDS 0.4 ml 5 N NaOH ------ 10 ml total | |
hight salt solution: 3 M NaOAc pH 5.2(same solution as used in precipitating DNA) | ||
Remarks:
Even if kept at -20° C DNA from this quality will degrade over a period of a year or so when not phenolized carefully (ca. 3 x)! Take care that no interphase remains.